Download TRANSVERSE SARCOMERE SPLITTING A Possible Means of

Published March 1, 1979
TRANSVERSE
SARCOMERE
SPLITTING
A P o s s i b l e M e a n s o f L o n g i t u d i n a l G r o w t h in C r a b M u s c l e s
S. S. J A H R O M I
and M I L T O N P. C H A R L T O N
From the Zoology Department, University of Toronto, Toronto, Ontario, Canada M5S 1A1. Dr.
Jahromi's present address is Biology Department, Pahlavi University, Shiraz, Iran.
ABSTRACT
KEY WORDS transverse sarcomere splitting
growth 9 muscle
myofilaments 9
ultrastructure
Increase in length of muscle fibers during the
growth of animals can involve both lengthening of
sarcomeres (I, 5, 6, 13, 15, 23) and addition of
sarcomeres (10, 13, 15, 16, 26). The mechanism
by which sarcomeres are added, however, still
remains unclear. A u b e r (5) suggested that Z disks
split transversely and that a new sarcomere grows
between the two Z-disk fragments during development of blowfly flight muscles. In vertebrate
muscle, the sarcomeres are somewhat shorter near
the ends of a muscle cell (13, 14) and protein
synthesis is greatest there (16, 26). This has led
numerous investigators (see reference 14) to suggest that sarcomeres are added at the ends of
muscle cells.
Nonuniform sarcomere lengths are known to
occur within single muscle cells in both vertebrate
and invertebrate muscles (2, 12, 13). As FranziniArmstrong (12) pointed out: "the possibility
should be explored that a continuous addition of
new sarcomeres underlies the noticeable variabil-
736
ity in A-band length of crustacean fibres."
In this paper we present ultrastructural evidence
of transverse splitting of sarcomeres which could
underly longitudinal growth as well as nonuniformity of sarcomere length within single muscle
cells in blue crabs.
MATERIALS AND METHODS
Blue crabs, Callinectes sapidus, were obtained from local
suppliers in Toronto and kept in tanks of three-quarter
strength artificial seawater at ~15~ The maxilliped
exopodites were removed, and the flagellum abductor
mu~les (muscles 78, 87, and 102 of reference 9) were
exposed by removing most of the overlying exoskeleton
on one side of th~ appendage. The flagellum joint was
immobilized and the muscles were fixed in situ at rest
length for 1 h in 2.5% glutaraldehyde containing 0.2%
formaldehyde and 0.15 M sodium cacodylate buffer at
pH 7.3. Muscles were then placed in cacodylate (0.15
M)-buffered sucrose (0.3 M) wash containing 0.06 M
NaCI and 2 mM CaC12 for 2-3 h (H. L. Atwood,
unpublished observations). Bundles of fibers were dissected from the exopodite and postfixed in 2% OsO4 (in
0.15 M cacodylate). Other procedures employed were
standard for this laboratory. (20).
J. CELL BIOLOGY9 The Rockefeller University Press. 0021-9525/79/03/0736-0751.00
Volume 80 March 1979 736-742
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Transversely split sarcomeres are seen in mouthpart muscles of the blue crab in
the electron microscope. Sarcomeres split only at the H zone. Two new
sarcomeres are formed by a Z disk which appears in the H zone of the splitting
sarcomere. Splitting may involve breaking of the thick filaments in the H zone,
elongation of these filaments, and formation of both new actin filaments and Zdisk materials. Sarcomere splitting would allow longitudinal growth of muscle
cells without lengthening of sarcomeres and concomitant changes in contractile
properties.
Published March 1, 1979
RESULTS
DISCUSSION
Mechanism of Splitting
We propose the following tentative scheme for
the mechanism of sarcomere splitting:
(a) Thick filaments are bisected at the H zone,
and the two halves are pulled toward opposite Z
lines.
(b) New myosin molecules are added to the
cut ends of the thick filaments. The heads of the
new myosin molecules point in the direction opposite to that of those in the existing half fdament
to which they become attached.
(c) Coincident with the assembly of new thick
filaments, cross bridges become available to which
actin molecules attach and subsequently form new
thin filaments. Thin filaments elongate.
(d) New Z line material appears in the split
and the new thin filaments are attached to it.
The exact mechanism and the sequence of these
steps are unknown. However, actin molecules in
the presence of myosin filaments can spontaneously self-assemble into contractile units, and Zline material is not needed for this assembly (11,
17). Because no partially split sa~omeres were
seen in which the split did not reach one edge of
the myofibril, we presume that splitting begins at
the edge of a myofibril and not in the middle. It is
not known what initiates or controls the splitting
process.
Origin o f Sarcomere Splitting
According to Tiegs (24, 25) and Ruska and
Edwards (22), the existence of verniers (regions
S. S. JAnROMIAND MILTONP. CrlAV,LXOr~
TransverseSarcomereSplitting
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The flagellum abductor muscle has short sarcomeres (2-4 tzm) which are usually well aligned
across several adjacent myofibrils. Some aspects
of the physiology of this muscle have been described by Burrows and Willows (7) and by Chadton (8). The contractions are fast and the muscle
is extremely fatigue resistant. The muscle is well
supplied with blood vessels, and each fiber has a
thick cortex of mitochondria. This muscle has
distinct myofibrils completely separated by sarcoplasmic reticulum elements and T tubules. Myofibrils are well separated into small groups by layers
of mitochondria. There is a distinct H zone in the
center of the A band.
When longitudinal sections are viewed in the
electron microscope, some sareomeres appear to
be partially split at the H zone to form two "new"
sarcomeres (Fig. 1). A new Z line forms the apex
of the split and is flanked by broken thick illaments in two short A bands whose combined total
length is greater than that of the unsplit A bands.
The I bands are shorter opposite the apex of the
split than at other unsplit regions of the sarcomere. In the partially split sarcomeres, two new
sarcomeres gradually taper into one sarcomere
and the H zone displays a Y shape pointing in the
direction of the split. In each new sarcomere, the
H zone is found closer to the apex Z line than to
the Z lines at the opposite ends of the new
sarcomeres. Evidently, the thin filaments attached
to the apex Z line must be quite short because the
H zone is close to the Z line in this region. In split
sarcomeres, the diads and triads are found at the
H zone as in normal sarcomeres of this muscle.
The splitting is sometimes confined to a single
sarcomere of one myofibril but can often extend
across several adjacent sarcomeres in a group of
myofibrils. In the latter case, the split sarcomeres
taper toward the apex of the split, and the H
zones with their diads or triads display the Y
shape. Occasionally, two short sarcomeres of
equal length are seen, presumably produced by
recent splitting (Fig. 2).
No partially split sarcomeres were seen in which
the split did not touch one edge of the sarcomere.
Within a group of myofibrils, splitting may occur
in the inner myofibrils and advance toward the
periphery, or vice versa (Fig. 3). Independent
splitting can occur on both sides of a group of
myofibrils, i.e., the apexes of splits can point in
opposite directions within one group of myofibrils
(Fig. 4).
Serial longitudinal sections were examined to
determine the appearance of splits at different
depths within a group of myofibrils. Fig. 4a and b
shows two splits on each of two serial sections
separated by a depth of 2.1 /~m. One split traversed more of the group of myofibrils while the
other split traversed less of the group in deeper
sections.
Splitting occurs randomly along the length of a
myofibril. For example, in one group of myofibrils
followed longitudinally on a single section for 132
sarcomeres, splits were observed at sarcomere
numbers 14, 22, 44, and 80. Splitting in one
group of myofibrils was not correlated with splitting in adjacent groups of myofibrils.
Published March 1, 1979
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FIGURE I Partial transverse sarcomere split seen in a longitudinal section. Thick filaments are broken
(arrows) in the region of the apex Z line (AZ). The new sarcomeres (NS) are formed between the apex Z
line and the immediately adjacent Z lines of the splitting sarcomere, H , H zone. • 38,100,
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Published March 1, 1979
in which n sarcomeres of one myofibril are apposed by n + 1 sarcomeres in the adjacent myofibril) can be explained by an underlying helicoidal
arrangement of the Z line. A section cut at an
angle through the axis of a Z helicoid would
produce a vernier appearance (22). The splitting
described in the present work bears a superficial
similarity to verniers but in our material n = 1
whereas verniers involve several sarcomeres. Occasionally, verniers in the maxilliped muscle do
involve sarcomere splits, but this is not always the
case. It is, of course, possible that the sarcomere
splitting could produce the verniers.
It is unlikely that split sarcomeres are related to
helicoidal muscle structure as described by Tiegs
(24, 25), Ruska and Edwards (22), or Peachey
and Eisenberg (21) since splitting only occurs at
H zones. If splitting were due to a regular helicoid
structure, the splits would occur at all sarcomere
zones.
FIGURE 2 A longitudinal section through a group of
myofibrils, showing a pair of short sarcomeres (arrows).
Except for the two Z lines delineating the short sarcomeres, the Z lines of neighboring myofibrils appear to
be nearly in register. • 10,400.
Why would muscle growth be accomplished by
sarcomere splitting? There is now ample evidence
that in crustacean skeletal muscle the speed of
contraction is inversely related to the sarcomere
length (3, 4, 18, 19). Therefore, the maintenance
of a particular mechanical function in a crustacean
muscle, barring other structural or biochemical
changes, is dependent on the maintenance of a
particular sarcomere length. The flagellum abductor is a fast muscle and has short sarcomeres.
Increases in the sarcomere length during longitudinal growth of the muscle cells would gradually
result in transformation of the fast cells into stow
cells. However, sarcomere splitting could prevent
such a transformation by reducing the sarcomere
length, thus retaining the fast activity of these
muscle cells. Obviously, the addition of new sarcomeres would render the lengthening of other
sarcomeres unnecessary during growth.
Sarcomere length could also be maintained
during growth if sarcomeres were added at the
ends of muscle cells (13, 14, 16, 26) or if new
sarcomeres were produced between the two halves
of split Z lines (5). The scheme presented in this
paper might have some advantage over the above
methods of sarcomere addition since splitting at
the H zone would leave a template (bisected
myosin filaments) which would guide the assembly
of the new sarcomeres.
The observation of sarcomere splitting is as yet
uncorrelated with actual growth of the muscle or
S. S. JAHROMI AND MIL'ION P, CHARLTON Transverse Sarcomere Splitting
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Sarcomere Splitting and Growth
Published March 1, 1979
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FIGURE 3 A longitudinal section through a group of myofibrils in which the inner myofibrils are split.
Splitting is incomplete in one sarcomere (arrow), and the split appears to progress toward the periphery.
Notice that in the myofibrils which have split, Z lines form a bulge around the split area. x 13,400.
of the whole animal. A t present there are two
different views in the literature regarding longitudinal growth of crustacean muscle. Bittner and
Traut (6) have reported that lengthening of muscle fibers in various crayfish skeletal muscles occurs by sarcomere lengthening, whereas Govind
et al. (15) have found that lengthening of limb
740
muscles in lobsters is accomplished in early stages
by sarcomere lengthening and in later stages by
sarcomere addition. The sarcomere splitting described herein could underly the addition of sarcomeres reported by Govind et al. (15). In crustaceans, increase in body size occurs after moulting of the exoskeleton. It would be interesting to
THE JOURNAL OF CELL BIOLOGY" VOLUME 80, 1979
Published March 1, 1979
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FIGURE 4 Serial l o n g i t u d i n a l sections t h r o u g h a g r o u p o f m y o f i b r i l s s h o w i n g t w o splitting r e g i o n s n i n e
s a r c o m e r e s a p a r t (S1 a n d $2 ), T h e t w o sections (a a n d b ) w e r e s e p a r a t e d b y 2.1 /zm. O n e split a r e a ( S 1 )
t r a v e r s e d m o r e o f the m y o f i b r i l s w h i l e the o t h e r split ( $ 2 ) t r a v e r s e d f e w e r o f the m y o f i b r i l s as the d e p t h o f
s e c t i o n i n g w a s i n c r e a s e d (a to b ) . • 3 , 2 0 0 B a r s , 1 /~m.
determine whether there is a higher incidence of
sarcomere splitting immediately before or after
moulting.
W e t h a n k I r e n e K w a n f o r t e c h n i c a l assistance. P r o f e s s o r
H . L . A t w o o d a n d D r . H . S i l v e r m a n kindly r e v i e w e d
the m a n u s c r i p t .
A f e l l o w s h i p to S. S. J a h r o m i f r o m the I r a n i a n
N a t i o n a l C o u n c i l f o r Scientific R e s e a r c h is a c k n o w l e d g e d . M . P. C h a r l t o n is a P o s t d o c t o r a l F e l l o w o f the
M u s c u l a r D y s t r o p h y A s s o c i a t i o n o f C a n a d a . T h i s res e a r c h w a s s u p p o r t e d b y g r a n t s to H . L . A t w o o d f r o m
the M u s c u l a r D y s t r o p h y A s s o c i a t i o n o f C a n a d a a n d the
National Research Council of Canada.
R e p r i n t r e q u e s t s s h o u l d be a d d r e s s e d to D r . C h a r l t o n .
Received for publication 18 July 1978, and in revised
f o r m 21 N o v e m b e r 1978.
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